Polymers containing quaternized nitrogen

ABSTRACT

The invention provides polymers, methods of preparing polymers, and compositions that include polymers, wherein said polymers include a plurality of two-carbon repeating units in a polymer chain, wherein one or more of the two-carbon repeating units of the polymer chain have tertiary amine or pyridine-containing substituents; and at least about 10% of the nitrogen atoms of the tertiary amine or pyridine-containing substituents are quaternized with alkyl groups or with an alkyl group that contains one or more ethylene glycol groups. The alkyl or ethoxylated alkyl groups can also be at least partially fluorinated. The polymers can be used to provide antimicrobial surfaces and antifouling coatings.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/507,361, filed Aug. 21, 2006, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 60/709,925, filedAug. 19, 2005, and is also a continuation-in-part of U.S. patentapplication Ser. No. 11/063,242, now U.S. Pat. No. 7,709,055, filed Feb.22, 2005, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/546,656, filed Feb. 20, 2004,which applications are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant NumbersN00014-02-1-0170 awarded by the Office of Naval Research and PP-1454awarded by the Strategic Environmental Research and Development Program(SERDP) of the Department of Defense. The United States Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Marine fouling is a major problem in the transport of materials by seaas it can raise fuel consumption by as much as 30%. Environmentallyfriendly coatings that protect the hulls of ships below the waterlineagainst fouling by seaweed, barnacles, and other organisms are currentlysought by the shipping industry. Fouling by these organisms producesadditional drag on the ship, thereby increasing operating andmaintenance costs.

Antifouling paints containing tin and copper biocides are currently usedbecause of their effectiveness against most forms of marine fouling.Many of these biocidal organometallic compounds are environmentallypersistent. They can cause damage to the ecosystem and enter the foodchain. The ban on tributyltin (TBT) antifoulants by the InternationalMaritime Organization will be effective in 2008, and copper-basedcoatings are expected to face similar restrictions in the near future.

Non-toxic “fouling-release” or “fouling-repellant” coatings are oneclass of alternatives to biocidal coatings. Silicone-based paints arecommercially available, but do not satisfy many desired performancecharacteristics. The soft silicones do not withstand the rigorousdemands of the marine environment, do not sufficiently and consistentlyself-clean, or, due to polymer restructuring or other degradationpathways, lose many of the desirable surface properties with time andexposure to marine organisms.

Several fouling release (FR) coating systems are commercially available,mostly based on silicone polymers, yet none meet all of the desiredperformance characteristics. Many commercially available coating systemslack the toughness required to withstand the rigorous physical demandsof the marine environment, do not sufficiently and consistentlyself-clean, and due to polymer restructuring or other degradationpathways, lose many of the desirable surface properties with time andexposure to the marine environment.

Current understanding of antifouling materials is that the mosteffective copper-free fouling control systems are low surface energycoatings, namely silicone or fluoropolymer based coatings that minimizethe adhesion strength between fouling organisms and surface. Forextended performance life, these coating systems should have controlledand stable surface energy, elastomeric properties, and should adherewell to the substrate.

What is needed is a material that has antimicrobial properties to reducemarine biofouling, thereby decreasing the accumulation and buildup ofmarine organisms and aiding in their removal. Such a material wouldpreferably lack toxic copper or tin metals, and lower the strength ofadhesion between marine fouling organisms and surfaces in contact with amarine environment. The diversity of fouling organisms and environmentalconditions worldwide makes the task of developing a coating that resistsfouling and/or self-cleans challenging, and novel solutions to theproblem are urgently needed.

SUMMARY

The invention provides a polymer that includes a plurality of two-carbonrepeating units in a polymer chain, wherein one or more of thetwo-carbon repeating units of the polymer chain have pyridine-containingsubstituents; and at least about 10% of the nitrogen atoms of thepyridine-containing substituents are quaternized with (C₁-C₃₀)alkylgroups or with an alkyl group that contains one or more ethylene glycolgroups.

The polymer of the invention can also include one or more polymer chainsubstituents selected from aryl groups, alkyl groups, and alkoxycarbonylgroups, wherein any alkyl, aryl, or alkoxy is optionally substitutedwith one or more alkyl, alkoxy, halo, dialkylamino, trifluoromethyl,ethylene glycol, or perfluoroalkyl groups.

The alkyl groups that are used to quaternized the pyridine-containingsubstituents can be at least partially fluorinated. The ethylene glycolgroups used to quaternized the pyridine-containing substituents can havealkyl groups in their chain that are also at least partiallyfluorinated. For example, at least about 10% of the (C₁-C₃₀)alkyl groupscan be at least partially fluorinated. In another embodiment, at leastabout 20% of the (C₁-C₃₀)alkyl groups are at least partiallyfluorinated. In other embodiments, at least about 50%, or about 80% toabout 99% of the (C₁-C₃₀)alkyl groups can be at least partiallyfluorinated.

The polymer can have at least about 50% of the nitrogen atoms of thepyridine-containing substituents quaternized with (C₁-C₃₀)alkyl groups.In polymers with fewer than 100 pyridine groups, the number ofquaternized pyridines or partially fluorinated alkyl groups as indicatedby any percentage limitation can be rounded up to the nearest integer.

One or more of the partially fluorinated (C₁-C₃₀)alkyl groups can be,for example, (C₆-C₁₀)perfluoroalkyl(C₃-C₁₀)alkyl groups. A specificexample of a (C₆-C₁₀)perfluoroalkyl-(C₃-C₁₀)alkyl groups is a6-perfluorooctyl-1-hexyl group.

The polymer can exhibits antifouling properties. Antifouling propertiesinclude antimicrobial and antialgal properties, such as activity againstmarine algae, algal spores, bacterial cells, diatoms, and protozoa.

The polymer can have a molecular weight of about 5 kDa to about 500 kDa.In some embodiments, the polymer can have a molecular weight of about 10kDa to about 150 kDa.

The invention also provides an antifouling surface that includes asurface coating comprising a polymer as described herein. Theantifouling surface can be prepared by coating a surface with a polymerof the invention. The antifouling surface can also include a base layerof a different polymer, for example, a plexiglass or an elastomericpolymer.

The invention further provides a polymer that includes at least onesegment of formula I:

wherein

each R¹ is independently aryl or alkoxycarbonyl;

each R² is independently a pyridine or a pyridine(C₁-C₁₀)alkyl group,wherein at least 10% of the nitrogen atoms of the pyridine moieties arequaternized with (C₁-C₃₀)alkyl groups or with an alkyl group thatcontains one or more ethylene glycol groups;

each R³ is independently hydrogen or methyl;

wherein any alkyl, aryl, or alkoxy is optionally substituted with one ormore alkyl, alkoxy, halo, dialkylamino, trifluoromethyl, ethyleneglycol, or perfluoroalkyl groups;

each m is about 5 to about 2000;

each n is about 5 to about 2,000;

p is about 5 to about 100; and

the dispersement of each individual m subunit and each individual nsubunit on either side of z* is random and each individual m subunit andeach individual n subunit occurs interchangeably with any other m or nsubunit within the brackets of formula I; or

the dispersement of each individual m subunit and each individual nsubunit on either side of z* is that of a block copolymer.

The dispersement of subunits on either side of z* indicates that thepolymer can be prepared by either anionic polymerization, free-radicalpolymerization, or any other technique known to those of skill in theart. The resulting polymer can thus be a random copolymer, terpolymer,etc., or the polymer can be a block polymer with two or more differenttypes of blocks.

In one embodiment, each R¹ can be phenyl or butoxycarbonyl, or acombination thereof. In one specific embodiment, each R¹ is phenyl andthe polymer is a block copolymer. In another embodiment, each R¹ isbutoxycarbonyl and the polymer is a random copolymer.

In one embodiment, each R² is 4-pyridine. Other pyridine substitutionscan also be used, for example, 2-pyridines and 3-pyridines. The pyridinegroups can be at least 20% quaternized with partially fluorinated(C₁-C₃₀)alkyl groups. The pyridine groups can also be at least 20%quaternized with non-fluorinated (C₁-C₃₀)alkyl groups.

At least about 10% of the (C₁-C₃₀)alkyl groups are at least partiallyfluorinated.

The values of m, n, and p can be selected such that the molecular weightof the polymer is about 5 kDa to about 500 kDa, about 10 kDa to about250 kDa, about 20 kDa to about 150 kDa, or about 40 kDa to about 100kDa.

The polymers can be employed to prepare an antifouling surface alone, orin combination with a base layer of another polymer, for example, aplexiglass or an elastomeric polymer.

The invention further provides a method of preparing an antimicrobialsurface that includes coating at least a portion of a surface with apolymer that has a plurality of two-carbon repeating units in a polymerchain, wherein one or more of the two-carbon repeating units of thepolymer chain have nitrogen-containing substituents;

the nitrogen-containing substituents comprise tertiary amines orpyridine groups; and (a) at least about 10% of the nitrogen atoms of thenitrogen-containing substituents are quaternized with (C₁-C₃₀)alkylgroups; or (b) at least about 10% of the nitrogen atoms of thenitrogen-containing substituents are quaternized with an alkyl groupthat contains one or more ethylene glycol groups; to provide theantimicrobial surface.

The polymer of the antimicrobial surface can have at least about 10% ofthe (C₁-C₃₀)alkyl groups can be at least partially fluorinated. In otherembodiments, at least about 50%, at least about 80%, or at least about90% of the (C₁-C₃₀)alkyl groups are at least partially fluorinated.

The invention further provides a block copolymer that includespolymerized 4-vinyl pyridine in a first block and styrene in a secondblock, wherein at least about 10% of the nitrogen atoms of the 4-vinylpyridine groups are quaternized with (C₁-C₃₀)alkyl groups, and whereinat least about 10% of the (C₁-C₃₀)alkyl groups are at least partiallyfluorinated.

In any polymer of the invention, the terminal groups of the polymer willbe determined by the method of polymerization used and the respectiveinitiator and quench used in the preparation process. One skilled in theart will readily understand the variety of terminal groups that can beprovided by the initiators and quenching agents. Typical end groupsinclude sec-butyl groups and phenyl groups. Other terminal groupsderived from the quenching agent include hydrogen, and various TEMPO andsilyl derivatives, for example, a dimethyl(2-perfluorooctyl)ethylsilylgroup.

The variables and limitations described for one general or specificembodiment for any polymer described herein can also be applied to otherembodiments, for example, other variations of the polymer of theinvention and variations of the embodiments provided in the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates C K-edge and N K-edge NEXAFS spectrum of apoly(4-vinylpyridine) surface obtained at an X-ray incident angle of 55°and entrance grid bias at the channeltron electron multiplier of −150 V.The dotted curve shows the exponential background that was subtracted inthe N K-edge region.

FIG. 2 illustrates IR spectra of (a) PS_(62k)P4VP_(66k); (b)PS_(62k)P4VP_(66k) quaternized with 1-bromohexane; and (c)PS_(62k)P4VP_(66k) reacted with 0.3 equivalents of F8H6Br followed by anexcess of H6Br. Preliminary peak assignments: 1640 cm⁻¹ C═N⁺ stretchingvibrations; 1200 cm⁻¹ to 1300 cm⁻¹ C—F stretching vibrations; 700 cm⁻¹styrene C—H bending vibrations.

FIG. 3 illustrates 300 MHz ¹H NMR spectra of (a) PS_(62k)P4VP_(66k) and(b) PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br in CDCl₃.

FIG. 4 illustrates C 1s (a) and N 1s (b) NEXAFS spectra of surfacesprepared by spin-coating poly(4-vinylpyridine) and poly(N-hexylpyridinium bromide) from chloroform solutions on silicon wafers; P4VPmolecular weight was 60 kDa. The surfaces were annealed for 12 hours ina vacuum oven at 120° C. The spectra were obtained at an X-ray incidentangle of 55° and the channeltron entrance grid bias of −150 V.

FIG. 5 illustrates C 1s (left) and N 1s (right) NEXAFS spectra ofPS-b-P4VP copolymers before (a and b) and after (c and d) quaternizationwith 1-bromohexane. The surfaces were prepared by spin coating 5% (w/v)solutions of the block copolymers in chloroform on silicon wafers andannealing at 150° C., above the T_(g) of the two blocks, for 12 hours invacuum. Glass transition temperatures (T_(g)) of PS and P4VP are about100° C. and 142° C., respectively (see Polymer 1998, 39, 2615-2620). TheNEXAFS spectra were obtained at an X-ray incident angle of 55°.

FIG. 6 illustrates C 1s (left) and N 1s (right) NEXAFS spectra ofspray-coated PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br

and PS_(62k)P4VP_(66k)H6Br (----) quaternized diblock copolymersurfaces. Spectra were obtained at 55° X-ray incident angle.

FIG. 7 illustrates C 1s (left) and N 1s (right) NEXAFS spectra ofpoly(4-vinylpyridine-ran-n-butylmethacrylate) surfaces.

FIG. 8 illustrates N 1s XPS peaks from the P4VP_(60k) andP4VP_(60k)H6Br. Both the peaks are normalized to unit area.

FIG. 9 illustrates C 1s XPS spectra of spray coated surfaces of (a)fluorinated pyridinium block copolymers, and (b) non-fluorinatedpyridinium block copolymers. The corresponding N 1s spectra are shown in(c) and (d). The carbon peaks were normalized such that the total areaunder each carbon peak was equal to unity. The nitrogen peaks werenormalized so that the area under the N 1s proportional to the number ofnitrogen atoms, relative to the number of carbon atoms.

FIG. 10 shows photographs of S. aureus colonies on 1 inch×1 inch regionsof test surfaces.

FIG. 11 illustrates (a) settlement of Ulva spores and (b) growth ofsporelings after 10 days, on PS/PI(10/12)F10H9 andPS/P4VP(11/21)F8H6Br-30% surfaces; PDMS and glass surfaces were used ascontrols;

=hydrophobic and

=hydrophilic.

FIG. 12 illustrates detachment of Navicula from non-polar blockcopolymer with semifluorinated side-chains, and polar quaternized 4-VPsurfaces with semifluorinated and alkyl side-chains;

=hydrophobic and

=hydrophilic.

FIG. 13 illustrates XPS survey scan (top) and high resolution C1s peaks(bottom) of the pyridinium block copolymer surface prepared byspray-coating onto SEBS covered glass slides.

FIG. 14 illustrates (a) C 1s and (b) N 1s NEXAFS spectra of quaternizedpoly(4-vinylpyridine) polymer surfaces.

FIG. 15 illustrates the settlement of Ulva spores on different surfaces.Each point is a mean from 90 counts on 3 replicate slides. Bars show 95%confidence limits.

FIG. 16 shows images of settled spores (a, b, and c) and 7 day oldsporelings (d, e, and f) on glass (a and d), PDMS (b and e), andP(4VP-r-BMA)_(300k)H6Br (c and f). Image width is approximately 500 μm.The spores used in these experiments were from a different batch thanthat used to obtain data in FIG. 17.

FIG. 17 illustrates detachment of Ulva sporelings from differentsurfaces plotted as % removal after 7 days growth. Coatings were exposedto a range of surface pressures using the water jet.

FIG. 18 illustrates settlement of Navicula on different surfaces. Eachpoint is the mean from 90 counts on 3 replicate slides. Bars show 95%confidence limits.

FIG. 19 illustrates detachment of Navicula from different surfaces. Eachpoint represents the mean percentage removal from 90 counts from 3replicate slides. Bars represent 95% confidence limits derived fromarcsine transformed data.

FIG. 20 illustrates various mers of various embodiments of the inventionwherein the mers can be selected in any combination in any order toprepare an antifouling polymer. The polymer merely illustrates thevariety of mers that can be used in various embodiments and is not anactual polymer that has been prepared.

DETAILED DESCRIPTION

The invention provides polymers that include substituents that containquaternized nitrogen atoms. The polymers exhibit antimicrobialproperties and can be used in antifouling coatings. The quaternizednitrogen groups are tethered to the polymer backbone and can inhibit orprevent the growth of microbes, such as bacterial colonies and/or algalcells, by effecting cell lysis and death. Specific organisms that can beinhibited include Ulva (green alga), Navicula (diatoms), andStaphylococcus aureus bacterium, among others.

The following definitions are used, unless otherwise described. Halo canbe fluoro, chloro, bromo, or iodo. Specific values listed below forradicals, substituents, and ranges, are for illustration only; they donot exclude other defined values or other values within defined rangesfor the radicals and substituents. The term “about” refers to a valuethat is greater than or less than the specified value by 5%, 10%, or25%. The term “about” can also refer to a value that is greater than orless than the specified value by one or two integers.

The phrase “one or more” is readily understood by one of skill in theart, particularly when read in context of its usage. For example, one ormore substituents on a phenyl ring refers to one to five, or one tofour, for example if the phenyl ring is disubstituted. One or moresubunits of a polymer can refer to about 5 to about 50,000, or anyincrement of about 100 or about 1,000 within that range. In otherembodiments, one or more refers to 1 to about 50, 1 to about 30, 1 toabout 20, 1 to about 12, 1 to about 10, 1 to about 8, 1 to about 5, 1 toabout 3, or 2.

It will be appreciated by those skilled in the art that compounds orpolymers of the invention having a chiral center may exist in and beisolated in optically active and racemic forms. Some compounds mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by synthesis from optically-active starting materials, by usingresolution of the racemic form by recrystallization techniques, bychiral synthesis, or by chromatographic separation using a chiralstationary phase). Thus, the compounds and polymers of this inventioninclude all stereochemical isomers arising from the various structuralvariations of these compounds.

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, etc., indicate that the embodiment describedmay include a particular feature, structure, or characteristic, butevery embodiment may not necessarily include that particular feature,structure, or characteristic. Moreover, such phrases are not necessarilyreferring to the same embodiment. Further, when a particular feature,structure, or characteristic is described in connection with anembodiment, it is submitted that it is within the knowledge of oneskilled in the art to affect such feature, structure, or characteristicin connection with other embodiments whether or not explicitlydescribed.

“Substituted” is intended to indicate that one or more hydrogens on agroup indicated in the expression using “substituted” is replaced with aselection from the indicated group(s), provided that the indicatedatom's normal valency is not exceeded, and that the substitution resultsin a stable compound. Suitable indicated groups include, e.g., alkyl,alkenyl, alkylidenyl, alkenylidenyl, alkoxy, aryloxy, halo, haloalkyl,hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl,alkanoyl, alkoxycarbonyl, aroyl, acyloxy, aroyloxy, amino, imino,alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy,carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl,alkylsulfonyl, cyano, acetamido, acetoxy, acetyl, benzamido,benzenesulfinyl, benzenesulfonamido, benzenesulfonyl,benzenesulfonylamino, benzoyl, benzoylamino, benzoyloxy, benzyl,benzyloxy, benzyloxycarbonyl, benzylthio, carbamoyl, carbamate, ethyleneglycol, isocyannato, sulfamoyl, sulfinamoyl, sulfino, sulfo, sulfoamino,thiosulfo, NR^(x)R^(y) and/or COOR^(x), wherein each R^(x) and R^(y) areindependently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle,cycloalkyl or hydroxy. As would be readily understood by one skilled inthe art, when a substituent is keto (i.e., ═O) or thioxo (i.e., ═S), orthe like, then two hydrogen atoms on the substituted atom are replaced.The substituent can be separated from the substituted atom by an alkylchain or ethylene glycol chain, and can be terminated by an alkyl group.

Specific values described for radicals, substituents, and ranges, aswell as specific embodiments of the invention described herein, are forillustration only; they do not exclude other defined values or othervalues within defined ranges, as would be recognized by one skilled inthe art.

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to about 30 carbon atoms,and often 1 to about 20, or 1 to about 12 carbon atoms. Examplesinclude, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl,1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,2-methyl-2-pentyl, 3-methyl-2-pentyl 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and3,3-dimethyl-2-butyl, hexyl, octyl, decyl, or dodecyl. The alkyl can beunsubstituted or substituted. The alkyl can also be optionally partiallyor fully unsaturated. As such, the recitation of an alkyl group includesboth alkenyl and alkynyl groups. The alkyl can be a monovalenthydrocarbon radical, as described and exemplified above, or it can be adivalent hydrocarbon radical (i.e., alkylene).

Alkoxy can be (C₁-C₁₂)alkoxy, such as, for example, methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,hexyloxy, or octyloxy. Any alkyl or alkoxy (including an“alkoxy”-carbonyl) can be optionally unsubstituted or substituted.

The term “aryl” refers to a monovalent aromatic hydrocarbon radical of6-20 carbon atoms derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,substituted benzene, naphthalene, anthracene, biphenyl, and the like.Aryl can also refer to an unsaturated aromatic carbocyclic group of from6 to 12 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed (fused) rings, wherein at least one ring is aromatic (e.g.,naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). The aryl can beunsubstituted or substituted.

As used herein, the term “mer” or “mers” refers to a unit of a polymerderived from a particular monomer. For example, a styrene mer refers toa segment of a polymer that was prepared from styrene to formpolystyrene, e.g., a phenethyl group wherein the ethyl group is a1,2-diradical. Accordingly, a mer can refer to a specific unit derivedfrom an unsaturated monomer, thus indicating, for example, a phenethyldiradical within the polymerized chain.

The term “ethoxylated” refers to a group that includes one or moreethoxy (—O—CH₂—CH₂—) groups, for example, about 2 to about 24 ethoxygroups, or about 3 to about 12 ethoxy groups. An ethoxylated group thathas two or more ethoxy or ethylene glycol groups refers to apolyethylene glycol, or PEG group. Ethoxylated or PEG groups can beterminated in an optionally substituted alkyl, for example, a methyl, orthey can terminate with hydrogen, e.g., a 2-hydroxyethoxy group.

The terms “semifluorinated” and “at least partially fluorinated” referto a group, for example an alkyl group, that has at least one hydrogenatom replaced by a fluorine atom. Semifluorinated groups include anycarbon chains, or carbon chains that are interrupted by one or moreheteroatoms (for example, oxygen), that contain one or more fluorineatoms. Typically the semifluorinated group will have one or more —CF₂—groups and can optionally terminate in a —CF₃ group. For example, thesemifluorinated group can be a group of the formula:

wherein each x is independently about 2 to about 20, and each y isindependently 0 to about 20. In other embodiments, x can be about 3 toabout 15, and y can be about 5 to about 15. Zonyl surfactants can beconsidered semifluorinated groups because a portion of the Zonylsurfactant group is a semifluorinated alkyl chain.

Zonyl® surfactants refer to ethoxylated fluoroalkyl chains with terminalalcohol groups. Zonyl® surfactants can be obtained from Dupont(Wilmington, Del.). These surfactants can be attached to appropriatelyfunctionalized block copolymers via the hydroxyl group, or via a halogroup which has replaced the hydroxyl group. Accordingly, polymershaving ethoxylated fluoroalkyl side chains can be prepared using anyappropriate Zonyl® surfactant. One example of a suitable Zonyl®surfactant that can be used to prepare ethoxylated fluoroalkyl sidechain-containing block copolymers is Zonyl FSO-100 [CAS # 122525-99-9].Other suitable Zonyl® surfactants include Zonyl FSN, Zonyl FSN-100, andZonyl FSO.

Ethoxylated fluoroalkyl groups that can be used in various embodimentsof the invention include moieties of formula Z:

wherein each q is independently 0 to about 25; each r is independently 0to about 18; and the moiety of formula Z is attached to a polymer chainsubstituent through an ester, amide, ketone, carbamate, or amine, or ispart of a quaternized nitrogen group.

When a nitrogen atom is quaternized, the resulting cation will beaccompanied by a corresponding anion. Typically the quaternization iscarried out by alkylating a nitrogen atom with an alkyl halide orsimilar halide, resulting in a halo counterion (anion). Typical haloanions include fluoro, chloro, bromo, and iodo, although bromo and iodoare of particular usefulness. The anions are not, however, limited tohalides and polymers with other anions can be prepared by one skilled inthe art.

As used herein, “contacting” refers to the act of touching, makingcontact, or of bringing within immediate proximity.

As used herein, “coating” refers to a manufacturing process orpreparation for applying an adherent layer to a workpiece or substrateweb. A coating can also be a layer of material that at least partiallycovers an underlying surface, such as a boat hull, pontoon, or any othersurface in need of an antifouling coating.

Methods of Making Compounds, Polymers, and Coatings

Processes for preparing the compounds and surface-active polymers of theinvention are provided as further embodiments of the invention. Relatedcompounds and compositions can be prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated inCompendium of Organic Synthetic Methods (John Wiley & Sons, New York),Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., AdvancedOrganic Chemistry, 3^(rd) Ed., (John Wiley & Sons, New York, 1985),Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency inModern Organic Chemistry, in 9 Volumes, Barry M. Trost, Ed.-in-Chief(Pergamon Press, New York, 1993 printing).

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will beabout −100° C. to about 200° C., solvents will be aprotic or proticdepending on the conditions required, and reaction times will be about 1minute to about 10 days. Work-up of standard organic transformationreactions typically consists of quenching any un-reacted reagentsfollowed by partition between a water/organic layer system (extraction)and separation of the layer containing the product. Work-up of reactionswith polymers typically consists of concentrating the reaction mixtureby removing a quantity of solvent, followed by precipitation of thepolymer using a solvent or solvent system in which the polymer has lowor substantially no solubility, such as, for example, methanol, or acombination of water and methanol.

The polymers of the invention can be synthetically modified, resultingin various substitutions on the mers of the backbone polymer. The merscan be substituted with side groups, such as, for example,semifluorinated (SF), poly(ethylene glycol) (PEG), or ethoxylatedfluoroalkyl side groups, or a combination thereof.

General and specific methods for preparing polymers, compositions, andcoatings are described in U.S. Patent Application Publication No.US-2006-0083854, which is incorporated herein by reference. The polymersof the invention can be used as protective coatings for surfaces in needof antifouling properties. The coatings can form single layer coatings,bi-layer coatings, or multi-layer coatings. The preparation of bi-layercoatings has been described by Ober et al., U.S. Pat. No. 6,750,296,which is incorporated herein by reference. These techniques can be usedto prepare bi-layer coatings that include the polymers described herein.

The invention also provides for a coating composition that includes apolymer that contains a quaternized nitrogen as described herein, incombination with other ingredients. Such other ingredients can include,for example, a polymer, water, one or more solvents, additives,stabilizers, colorants, dispersants, or combinations thereof.

The invention also provides a method of at least partially coating asurface by contacting the surface with a composition containing apolymer that contains a quaternized nitrogen as described herein. Thecoating procedure can be performed by brushing, immersing, pouring,solvent-casting, spin-coating, or spray-coating to contact the surfacewith the composition. Accordingly, the invention provides a method ofcoating or protecting a substrate, for example, a boat hull, frombiofouling.

The surface coated by the composition can be a layer of a thermoplasticpolymer. The thermoplastic polymer can at least partially covers a boathull, pontoon, or any other structure in need of such a coating. Theaverage-weight molecular weight of a polystyrene block of the a polymerthat contains a quaternized nitrogen as described herein in thecomposition used to coat the thermoplastic polymer can be within about20%, or about 10%, or about 5% of the average-weight molecular weight ofthe polystyrene block or blocks of a polymer comprising thethermoplastic polymer.

A bilayer can be formed and the bilayer can be annealed at a temperatureabove the glass transition temperature of the polystyrene blocks of thepolymers in the bilayer. The coating can result in a top layer of about30 nm to about 500 μm in thickness. Specifically, the coating can resultin a top layer of about 40 nm to about 150 μm in thickness. Morespecifically, the coating can result in a top layer of about 50 nm toabout 25 μm in thickness.

The polymers of the invention can provide a polymer that has anappropriate hydrophilic-lipophilic balance required for cell-membranedisruption effect, thus providing an antifouling coating when used tocoat marine surfaces. The polymers can provide a surface chemicalcomposition so that the pyridinium rings (of higher surface energy) arepresent at sufficient concentrations to latch on to the cell membranes,typically within the outer one to three nm of the surface. The polymerscan provide a sufficiently hydrophilic surface to prevent adsorption ofextracellular matrices of settling organisms. These extracellularmatrices can include proteins, glycoproteins, peptidoglycans, andnucleic acids that the organisms secrete for adhesion.

The amphiphilic nature of the ethoxylated fluoroalkyl chains of thepolymers minimize the adhesion strength of marine organisms. Otherpolymer chain substituents can provide effective screening of thepositively charged pyridinium rings so that the surface does not inducemicrobial settlement. Many embodiments are readily soluble commonsolvents such as THF, methanol, methylene chloride, chloroform,nitromethane, and nitrobenzene. The polymers have good film formingproperties. The polymers can have minimal or no solubility or swellingin water for under-water applications, and minimal or no toxicity tohuman cells.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the present invention could be practiced. It should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

EXAMPLES

Materials: Styrene (CAS no. 100-42-5, FW 104.15, >99%, Sigma-Aldrich)was stirred over dry di-n-butylmagnesium (received from Sigma-Aldrich asa 1.0 M solution in heptane), and 4-vinylpyridine (CAS no. 100-43-6, FW105.14, 95%, Aldrich) was stirred over calcium hydride (CAS no.7789-78-8, 90-95%, Aldrich) for 12 hours and distilled under vacuumafter three freeze-thaw-degas cycles.

Tetrahydrofuran (99.9%, Fisher) was distilled from Na/benzophenone.sec-Butyllithium (s-BuLi, CAS no. 598-30-1, CH₃CH₂CH(Li)CH₃, 1.4 Msolution in cyclohexane, Aldrich), lithium chloride (CAS no. 7447-41-8,LiCl, FW 42.39, 99.9%, Mallinckrodt),(heptadecafluoro-1,1,2,2-tetrahydrodecyl)-dimethylchlorosilane (CAS no.74612-30-9, F(CF₂)₈(CH₂)₂Si(CH₃)₂Cl, FW 540.72, >95%, Gelest),perfluorooctyl iodide (CAS no. 507-63-1, F(CF₂)₈I, FW 545.96, >98%,Fluka), 5-hexen-1-ol (CAS no. 821-41-0, HOCH₂(CH₂)₃CH═CH₂, FW 100.16,99%, Aldrich), 2,2′-azobisisobutyronitrile (CAS no. 78-67-1,N≡CC(CH₃)₂N═NC(CH₃)₂C≡N, FW 164.21, 98%, Aldrich), tributyltin hydride(CAS no. 688-73-3, (n-Bu)₃SnH, FW 291.06, 97%, Aldrich), carbontetrabromide (CAS no. 558-13-4, CBr₄, FW 331.63, 99%, Aldrich),triphenylphosphine (CAS no. 603-35-0, (C₆H₅)₃P, FW 262.29, 99%,Aldrich), and 1-bromohexane (CAS no. 111-25-1, CH₃(CH₂)₅Br, FW 165.07,98%, Aldrich) were used as received.

Poly(4-vinylpyridine-ran-butyl methacrylate) with a weight-averagemolecular weight of 300 kDa and 10 wt % of n-butyl methacrylate,anhydrous methylene chloride, N,N-dimethylformamide (DMF) andnitromethane were obtained from Aldrich and used without furtherpurification.

Polystyrene-block-poly(ethylene-co-butylene)-block-polystyrene (SEBS)triblock thermoplastic elastomer (Kraton G1652) was from KRATONPolymers. The solvents, methanol, chloroform, toluene, diethyl etherwere purchased from Fisher and used as received.

Example 1 Preparation of Polymers Containing Pyridine or PyridiniumGroups

Synthesis of polystyrene-block-poly(4-vinylpyridine) by anionicpolymerization. Polystyrene-block-poly(4-vinylpyridine) were preparedfollowing a literature procedure (Förster et al. J. Chem. Phys. 1996,104, 9956-9970), as illustrated in Scheme 1.

Polymerization was carried out in tetrahydrofuran at −78° C. usingsec-butyllithium initiator. Styrene was stirred with dibutyl magnesium,and 4-vinylpyridine was dried over calcium hydride before distillationunder vacuum. Tetrahydrofuran was refluxed over sodium/benzophenonecomplex and collected in a reaction flask containing lithium chloride(about 5 times the molar amount of sec-BuLi) by distillation.

The initiator (1.4 M solution in cyclohexane) was then injected,followed by the addition of styrene using a cannula. A small amount ofthe polymer solution was withdrawn from the flask after 45 minutes formolecular weight determination, and was terminated with anhydrous,oxygen-free methanol.

The 4-vinylpyridine monomer was then added to the reaction flask, atwhich point the color of the solution changed from orange to yellow.After 2 hours of polymerization at −78° C.,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane (10 timesmolar excess) was injected to terminate the polymer chains. The solutionwas slowly warmed to about 30° C., at which time a loss of color,signifying termination of the anions, was observed. The final polymercontent of the solution was about 5% (w/v).

The monomer conversion, determined from the masses of the monomers addedand the mass of the polymer obtained, was close to 100%. Molecularweight of the PS block was determined by gel permeation chromatography(GPC) of the polymer in THF using four Waters Styragel HT columnsoperating at 40° C., and Waters 490 ultraviolet (λ=254 nm) and Waters410 refractive index detectors. GPC indicated a narrow distribution withthe ratio of weight average molecular weight to the number averagemolecular weight less than 1.1. The molecular weight of the4-vinylpyridine block was obtained from the mass of added4-vinylpyridine and the PS molecular weight.

Two different diblock copolymers were prepared: one with PS and P4VPblock molecular weights of 11 kDa and 21 kDa, respectively, designatedas PS_(11k)P4VP_(21k), and the other with PS and P4VP block molecularweights of 62 kDa and 66 kDa, respectively, designated asPS_(62k)P4VP_(66k). The PS-b-P4VP copolymers were quaternized with6-perfluorooctyl-1-bromohexane (F8H6Br) (4) and 1-bromohexane (H6Br) toobtain block copolymers with semifluorinated side chains (6), asillustrated below in Scheme 4.

Synthesis of semifluorinated alcohol 3. 6-Perfluorooctyl-1-hexanol (CASno. 129794-54-3, F(CF₂)₈(CH₂)₆OH, FW 520.23) was prepared as describedin Höpken (Höpken et al. New Polymeric. Mater. 1991, 2, 339-356), and asillustrated in Scheme 2 below.

Perfluorooctyl iodide (40 g, 73 mmol) and 11 g of 5-hexen-1-ol (109.5mmol) were heated to 80° C. in a three-neck round-bottom flask fittedwith a reflux condenser and purged with nitrogen. About 200 mg (1.22mmol) of AIBN was added in 4 portions over a period of 6 hours, and thereaction mixture was maintained at 80° C. for a further 6 hours. Excess5-hexen-1-ol was removed by distillation (bp 56° C. at 11 mmHg).Reduction of iodo-adduct 2 was performed at 80° C. for about 24 hours byadding 30 mL of anhydrous toluene, 31.9 g (109.5 mmol) oftributyltinhydride, and 0.657 g (4 mmol) of AIBN. The product, whichsolidified on cooling, was separated by filtration and washed withtoluene to remove the tin dimer, (n-Bu)₃Sn—Sn(n-Bu)₃. The yield wasabout 60%.

Synthesis of semifluorinated alkyl bromide 4.6-Perfluorooctyl-1-bromohexane (CAS no. 195247-87-1, F(CF₂)₈(CH₂)₆Br, FW583.12) was synthesized following the procedure of Wang and Ober (LiquidCrystals 1999, 26, 637-648; Macromolecules 1997, 30, 7560-7567), and asillustrated below in Scheme 3.

Three grams (5.77 mmol) of 6-perfluorooctyl-1-hexanol and 3 g (9.05mmol) of CBr₄ were dissolved in a mixture of 6 mL of anhydrous THF and12 mL of anhydrous methylene chloride, and the solution was cooled to−5° C. Triphenylphosphine (2.37 g, 9.05 mmol) of was added in smallportions over a period of 15 minutes. After stirring for 1 hour at −5°C. and 6 hours at room temperature, the solvents were evaporated fromthe reaction mixture under vacuum and about 50 mL diethyl ether wasadded. An insoluble solid (triphenylphosphine oxide byproduct) wasseparated by filtration and the filtrate was concentrated to obtain thecrude product, which was purified by passing through a short silica gelcolumn with diethyl ether as the elution solvent. The yield was about85%.

Quaternization of PS-b-P4VP using 1-bromohexane. The PS-b-P4VP polymerwas reacted with about 5× moles of 1-bromohexane in anhydrous DMF at 80°C. for about 24 hours under nitrogen. Thus, 1.5 g (7.36 mmol 4-VP) ofthe PS_(62k)P4VP_(66k) diblock copolymer was dissolved in 10 mL ofanhydrous DMF and the reaction flask was purged with dry nitrogen forabout 15 minutes. Five milliliters of 1-bromohexane (35.6 mmol) wasadded and the reaction mixture was heated under nitrogen at 80° C. Thesolution turned dark green within about 2 hours of reaction. After 24hours, the reaction mixture was cooled to room temperature, and addeddrop-wise to 200 mL of diethyl ether at 0° C. resulting in a brownprecipitate of the polymer (polymer 5 in Scheme 4 below). The solid wasdissolved in chloroform, re-precipitated in diethyl ether, and driedunder vacuum.

Quaternization of PS-b-P4VP using 6-perfluorooctyl-1-bromohexane.

Quaternization of PS-b-P4VP using 6-perfluorooctyl-1-bromohexane isillustrated below in Scheme 4.

The PS-b-P4VP polymer was reacted with 0.3 equiv of6-perfluorooctyl-1-bromohexane in anhydrous DMF at 80° C. for about 24hours under nitrogen. The remaining pyridine groups were furtheralkylated using an excess of 1-bromohexane at 80° C. for 24 hours. Thereactions were carried out sequentially without isolation of thepartially quaternized block copolymer 5. Thus, 1 g (4.92 mmol 4-VP) ofthe PS_(62k)P4VP_(66k) diblock copolymer and 0.8630 g (1.48 mmol) of6-perfluorooctyl-1-bromohexane were dissolved in 10 mL of anhydrous DMFand heated to 80° C. under nitrogen for 24 hours, after which 5 mL (35.6mmol) of 1-bromohexane was added and the reaction continued for 24 hoursat 80° C. After cooling to room temperature the polymer was precipitatedin diethyl ether at 0° C. to obtain the partially fluorinated polymer 6shown in Scheme 4. It was further purified by re-precipitation from a20% (w/v) solution in chloroform into at least 20 fold volumetric excessof diethyl ether (0° C.) to obtain a fine green precipitate.

Quaternization of P4VP homopolymer and P(4VP-co-BMA) random copolymerusing 1-bromohexane. P4VP (2.5 g) with a molecular weight of 60 kDa, wasreacted with 4.3 g of 1-bromohexane (10% molar excess) in 25 g ofnitromethane at 80° C. for about 2 days. The color of the solutionchanged from bright green to dark green and finally brown. After coolingto room temperature, the viscous solution was poured into diethyl etherat 0° C. to obtain the quaternized polymer as a brown precipitate.Poly(N-hexyl-4-vinylpyridine-ran-n-butyl methacrylate) random copolymerwas similarly prepared by reacting 6 g of P(4VP-co-BMA), with an averagemolecular weight of 300 kDa, with 8 mL of 1-bromohexane in 60 mL ofnitromethane at 80° C. for 2 days, followed by precipitation of thepolymer in diethyl ether.

Results and Discussion: The PS-b-P4VP polymers were easily soluble indimethyl formamide. The use of DMF as a solvent resulted in a highdegrees of quaternization within shorter reaction times compared tochloroform, possibly due to higher reaction temperatures that could beused under non-pressurized conditions. Moreover, the PS_(11k)P4VP_(21k)formed a cloudy solution in chloroform, suggesting micelle formation.

The pyridinium block copolymer prepared by reacting 0.3 equiv of6-perfluorooctyl-1-bromohexane with the PS_(62k)P4VP_(66k) blockcopolymer, is denoted by PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br. The polymerPS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br was similarly prepared by reactingPS_(11k)P4VP_(21k) with 0.3 equivalents of F8H6Br followed by an excessof H6Br. The PS_(11k)P4VP_(21k) and PS_(62k)P4VP_(66k) block copolymersquaternized with 1-bromohexane alone are denoted byPS_(11k)P4VP_(21k)H6Br and PS_(62k)P4VP_(66k)H6Br, respectively. Thesewere readily soluble in chloroform or chloroform/methanol mixtures toform clear solutions or cloudy micellar dispersions.

All the block copolymers were end-capped with perfluorooctyl groups,however the PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br andPS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br polymers containing the F8H6Br sidechains are referred to herein as “fluorinated”, whereas thePS_(62k)P4VP_(66k)H6Br and PS_(11k)P4VP_(21k)H6Br polymers withoutsemifluorinated side chains will be referred to as “non-fluorinated”.

Initial polymer characterization. Infrared spectra of the polymers wereacquired using a Mattson 2020 Galaxy Series FTIR spectrometer. Polymerfilms for the IR spectroscopy were prepared on salt plates (KBr or NaCl)by drying solutions of the polymers in chloroform. ¹H and ¹⁹F NMRspectra were recorded using Varian Gemini spectrometer. CDCl₃ containing0.05% (v/v) tetramethylsilane was used as the solvent. Differentialscanning calorimetry was performed using a TA Instruments Q1000 seriesDifferential Scanning Calorimeter under nitrogen atmosphere. About 5 mgof sample was used with heating and cooling rates of 10° C./minutes.

Example 2 Characterization, Testing, and Evaluation of Polymer Coatings

The role of surface charge density on antibacterial activity has beenrecognized in some studies. For example, see Tiller et al. Biotechnol.Bioeng. 2002, 79, 465-471; and Kügler et al. Microbiology 2005, 151,1341-1348. Bacterial death occurred only above a threshold value ofsurface charge density. The experiments described herein using surfacesof poly(4-vinylpyridine) block copolymers showed that besides the lengthof the pyridinium block, it is the number of pyridinium rings in the topfew nanometers of the surface that determines the bactericidal activity.P4VP with a molecular weight of around 21 kDa was found to exhibitalmost 100% bactericidal effect against S. aureus. Moreover, a surfacewherein the pyridinium rings were densely covered by the alkyl sidegroups was found to be less effective than one in which the pyridiniumrings were exposed. Near-edge X-ray absorption fine structure (NEXAFS)spectroscopy and X-ray photoelectron spectroscopy (XPS), which candetermine the chemical composition within top 2 to 3 nm of a surface,were used in conjunction with contact angle measurements to study theeffect of surface chemistry on bactericidal activity.

Preparation of surfaces for antibacterial tests. Surfaces for bacterialassays were prepared on 3 inch×1 inch glass microscope slides. Toimprove adhesion of the pyridinium polymers to glass,polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (KratonSEBS G1652) was first spin-coated on the glass slides using a 10% (w/v)solution in toluene and annealed in a vacuum oven at 120° C. for 12hours. Solutions of the quaternized polymers were then sprayed on theSEBS-coated glass slides (heated to 80° C. on a hot-plate) using aBadger Model 250 airbrush (50 psi nitrogen gas pressure). Samples forNEXAFS and XPS analyses were also prepared by spin coating polymersolutions, typically 3 to 5% (w/v) solutions in chloroform, on siliconwafers using a Cee model 100CB spin coater at 2000 rpm (acceleration of1000 rpm/s) for 30 seconds.

Contact Angle and Surface Roughness. Contact angles were measured usinga NRL contact angle goniometer (Ramé-Hart Model 100-00) at roomtemperature. Dynamic water contact angle measurements were performed byaddition and retraction of a drop of water on the surface. Surfaceroughness was determined using a 3-D interferometric non-contact surfaceprofiler (ADE Phase-Shift MicroAXM-100HR). Root-mean-square (rms)roughness values were determined over regions of 631 μm×849 μm size andaveraged over at least 10 measurements.

NEXAFS Spectroscopy. NEXAFS experiments were carried out on the U7ANIST/Dow materials characterization end-station at the NationalSynchrotron Light Source at Brookhaven National Laboratory. The X-raybeam was elliptically polarized (polarization factor=0.85), with theelectric field vector dominantly in the plane of the storage ring. Thephoton flux was about 1×10¹¹ photons/s at a typical storage ring currentof 500 mA. A spherical grating monochromator was used to obtainmonochromatic soft X-rays at an energy resolution of 0.2 eV. C and NK-shell NEXAFS spectra were acquired for incident photon energy in therange 270 eV to 440 eV. A computer controlled goniometer, to which thesample holder was attached, was used to vary the orientation of thesample with respect to the X-ray beam. The partial-electron-yield (PEY)signal was collected using a channeltron electron multiplier with anadjustable entrance grid bias (EGB). All data reported in this Exampleare for a grid bias of −150 V.

The channeltron PEY detector was positioned at an angle of 45° withrespect to the incoming X-ray beam, and in the equatorial plane of thesample chamber. To eliminate the effect of incident beam intensityfluctuations and monochromator absorption features, the PEY signals werenormalized by the incident beam intensity obtained from the photo yieldof a “dirty” gold grid (Stöhr J. NEXAFS Spectroscopy. Springer-Verlag:New York, 1996; Chapter 5, p 114). A linear pre-edge baseline wassubtracted from the normalized spectra, and the edge jump wasarbitrarily set to unity at 320 eV, far above the C K-edge, a procedurethat enabled comparison of different NEXAFS spectra for the same numberof carbon atoms.

The N 1s Auger PEY was determined by subtracting the exponentiallydecreasing background arising from C atoms in the region between 390 eVand 430 eV, as shown in FIG. 1. Energy calibration was done using ahighly oriented pyrolytic graphite (HOPG) reference sample. The HOPG 1sto π* transition was assigned an energy of 285.5 eV according to theliterature value (Rosenberg et al. Phys. Rev. B 1986, 33, 4034-4037).The simultaneous measurement of a graphite-coated gold grid allowed thecalibration of the photon energy with respect to the HOPG sample. Theerror in the energy calibration is expected to be within ±0.5 eV. Eachmeasurement was taken on a fresh spot of the sample in order to minimizepossible beam damage effects. Charge compensation was carried out bydirecting low energy electrons from an electron gun onto the samplesurface.

X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS)measurements were performed using a Kratos Axis Ultra Spectrometer(Kratos Analytical, Manchester, UK) with a monochromatic Al Kα X-raysource (1486.6 eV) operating at 225 W under 1.0×10⁻⁸ torr. Chargecompensation was carried out by injection of low energy electrons intothe magnetic lens of the electron spectrometer. The pass energy of theanalyzer was set at 40 eV for high resolution spectra and 80 eV forsurvey scans, with energy resolutions of 0.05 eV and 1 eV, respectively.The spectra were analyzed using CasaXPS v. 2.3.12Dev4 software. The C—Cpeak at 285 eV was used as the reference for binding energy calibration.

Antibacterial tests. Viable counts. Five milliliters of Trypticase SoyBroth (TSB; per liter: 17 g casein peptone, 3 g soy meal peptone, 2.5 gD(+)glucose, 5 g NaCl and 2.5 g dipotassium hydrogen phosphate) wasinoculated with 100 μL of an overnight culture of S. aureus, andincubated at 37° C. for 4 hours. The cells were centrifuged at 5000 rpm(room temperature) for 1 minute using an Eppendorf model 5415 Cmicrocentrifuge, and the pellet was re-suspended in 1 mL sterilefiltered water. The suspension of S. aureus (˜10⁶ cells/mL) was sprayedon test surfaces. Sprayed surfaces were dried in air for about 2 minutesand then placed in a sterile petri dish. Molten agar-containing TSB(1.5% w/v of agar) was poured on the slides and allowed to solidify.Plates were incubated at 37° C. overnight (approximately 12 hours). Thenumber of bacterial colonies on the slide surface was counted using acolony counter. Three replicates per polymer were used. Corning glassslides that were not coated with the quaternized polymers were used ascontrols. Typical density of colonies on the glass controls were 100-150per cm² of the surface.

LIVE/DEAD® BacLight™ Bacterial Viability Assay. LIVE/DEAD® BacterialViability Kit (BacLight™) was obtained from Molecular Probes Inc. Equalvolumes of SYTO® 9 and propidium iodide (received as a solution inanhydrous dimethylsulfoxide) were mixed thoroughly in a microcentrifugetube. A suspension of S. aureus (˜10⁵ cells/mL) was prepared asdescribed above. The BacLight dye mixture (30 μL) was added to 1 mL ofthe cell suspension, which was then sprayed on the test surfaces.Immediately after spraying, the test surfaces were covered with glasscoverslips and incubated in the dark for 15 minutes. Phase-contrast andfluorescence microscopy were performed, within 30 minutes afterspraying, using an Olympus BX61 epifluorescence microscope with a 100×UPlanApo (N.A. 135) objective. The microscope was equipped with filtercubes for viewing SYTO® 9 and propidium iodide fluorescence. Images wereacquired using a Cooke SensiCam with a Sony Interline chip and Slidebooksoftware (Intelligent Imaging Inc.). Glass microscope slides were usedas controls.

Results and Discussion:

Polymer characterization. In order to interpret the NEXAFS spectra ofthe block copolymer surfaces, a quaternized homopolymer of4-vinylpyridine was prepared and used as a reference. P4VP with amolecular weight of 60 kDa was alkylated using 1-bromohexane. Unlike theblock copolymers that were not soluble in nitromethane, thequaternization reaction of the P4VP homopolymer could be performed inthis solvent. The resulting polymer is denoted by P4VP_(60k)H6Br.

A random copolymer of 4-vinylpyridine and n-butyl methacrylate (BMA)with 10 wt. % BMA and a total weight-average molecular weight of 300 kDawas alkylated with 1-bromohexane. The resulting high molecular weightcopolymer, denoted by P(4VP-r-BMA)_(300k)H6Br, was compared with thequaternized PS-b-P4VP diblock copolymers for antibacterial activity.Surfaces prepared using P(4VP-r-BMA)_(300k)H6Br were found to retainclarity when immersed under water, whereas the P4VP_(60k)H6Br polymerclouded upon water immersion.

FIG. 2 shows the IR spectra of the PS_(62k)P4VP_(66k) diblockcopolymers. The peak at 700 cm⁻¹, arising due to C—H bending vibrationsof the styrene phenyl ring, is unique to the PS block and is absent inpoly(4-vinylpyridine) homopolymers. The quaternization reaction resultedin an almost complete shift of the peak at about 1600 cm⁻¹,corresponding to the C═N stretching vibration of the pyridine ring, toabout 1640 cm⁻¹. The PS_(11k)P4VP_(21k) polymers showed a similar shiftof the peak at 1600 cm⁻¹ to 1640 cm⁻¹. Thus, the non-fluorinated as wellas the fluorinated pyridinium diblock copolymers showed a high degree ofquaternization, which was also evident from the XPS spectra of thesepolymers, as discussed in the NEXAFS Spectroscopy section below. Theextent of alkylation of P4VP is usually determined using the relativeintensities of the peaks at 1600 and 1640 cm⁻¹ (Panov et al. J. Appl.Spectr. 1975, 23, 958-962). However, in the case of the quaternizeddiblock copolymers, the PS block is also expected to show an aromaticC═C stretching resonance at 1600 cm⁻¹. Interestingly, as seen in FIG. 2,the 1600 cm⁻¹ peak expected for PS is highly suppressed in thequaternized block copolymer, while the aromatic C—H bending resonance ofPS at 700 cm⁻¹ is quite pronounced.

The expected polymer composition was further confirmed by ¹H and ¹⁹F NMRspectroscopy. FIG. 3 shows the ¹H NMR spectra of the PS_(62k)P4VP_(66k)block copolymer precursor and the PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Brfluorinated pyridinium polymer. The peak near 8.3δ in the spectrum ofthe un-quaternized polymer (FIG. 3 a) corresponds to the protons of thepyridine ring ortho to the nitrogen atom. The peaks near 6.4δ and 7.1δresult from the meta protons, and the protons of the styrene phenylrings.

The effect of quaternization is clearly evident in the ¹H NMR spectrumof FIG. 3 b, where the protons of the pyridine ring now appear at 8.2δand 9.1δ. These ¹H nuclei are less shielded due to the positive chargeon the carbon atoms of the ring, and thus appear at higher resonancefrequencies (or chemical shifts, δ). The positions of the phenyl ringprotons remain unchanged. Also seen are the protons of the alkyl sidechains, near 0.88δ and 2.7δ, the former attached to carbon atoms awayfrom the pyridinium ring while the latter attached to carbon atomscloser to the pyridinium ring. The peak near 2.1δ is likely from the—CF₂CH₂— protons of the semifluorinated alkyl side chains (see LiquidCrystals 1999, 26, 637-648). The 376.13 MHz ¹⁹F NMR spectrum ofPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br showed peaks at −81.4δ (—CF₃),−114.9δ (—CF₂CH₂—), −122.5δ, −123.4 δ, −124.1δ and −126.7δ (—CF₂CF₃).

Infrared spectroscopy of the pyridinium homopolymer, P4VP_(60k)H6Br,showed a nearly complete shift in the position of the C═N stretchingresonance from 1600 cm⁻¹ to 1640 cm⁻¹ indicating a high degree ofalkylation of the P4VP polymer. Using the peak at 1720 cm⁻¹corresponding to C═O stretching vibrations of n-butyl methacrylate as aninternal standard, the degree of quaternization in P(4VP-r-BMA)H6Br wasdetermined by the percent decrease in the absorbance of the C═Nstretching peak at 1600 cm⁻¹, and was found to be about 94%.

Unlike the semifluorinated alkyl side-chain ionenes (polymers withquaternary nitrogen atoms in the main chains) studied by Wang and Ober(Macromolecules 1997, 30, 7560-7567), or the 4-vinylpyridine polymersquaternized with ω-alkylphenylbenzoate derivatives studied by Masson etal. (Macromol. Chem. Phys. 1999, 200, 616-620), the pyridinium blockcopolymers did not show strong thermal transitions by DSC, possiblybecause of the relatively low density of the semifluorinated alkylgroups along the polymer backbone.

Preparation of test surfaces. The coating formulations are given inTable 1. Glass microscope slides, which were spin-coated with a layer ofSEBS, were used as substrates. The SEBS film results in an elastomericsurface in which the cylindrical domains formed by the PS end-blocks(˜7.5 kDa mol. wt.) act as physical crosslinks in a matrix of thepoly(ethylene-ran-butylene) central block (˜35 kDa mol. wt.). About 3 mgof the pyridinium polymer was used per cm² of the surface. Thespray-coated surfaces were dried in a vacuum oven at 60° C. for 24hours. The properties of the surfaces, characterized by contact anglemeasurements and NEXAFS spectroscopy, were found to be fairly sensitiveto the coating formulation and processing.

TABLE 1 Solutions used to prepared surfaces for antibacterial tests.Solution Polymer Formulation appearance PS_(11k)P4VP_(21k)H6Br* 1.5%(w/v)^(§) in 1:1 (v/v) Clear, pale chloroform-methanol blend yellowPS_(62k)P4VP_(66k)H6Br* 1.5% (w/v) in chloroform CloudyPS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br^(†) 1.5% (w/v) in 1:1 (v/v) Clear,dark chloroform-methanol blend greenPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br^(†) 1.5% (w/v) in chloroform Clear,dark green P(4VP-r-BMA)_(300k)kH6Br* 1.5% (w/v) in chloroform Clear,pale yellow *P4VP precursors quaternized using a molar excess of1-bromohexane alone. ^(†)P4VP precursors reacted with 0.3 equivalents of6-perfluorooctyl-1-bromohexane followed by reactions with an excess of1-bromohexane. ^(§)1.5% (w/v) = 0.015 g/mL.

Water Contact Angle Measurements. Table 2 lists the advancing andreceding water contact angles (CA), denoted by θ_(w,adv) and θ_(w,rec),respectively, on the spray-coated surfaces used in the antibacterialtests. The variation in the measured values was within ±2° and thevalues reported are averages of at least 5 measurements. The rmsroughness values, determined by optical interferometry, were close to 1μm—about 0.6 μm for PS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br, 0.9 μm forPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br, and 1.1 μm for P(4VP-r-BMA)H6Br. Anun-quaternized PS-b-P4VP copolymer, spin-coated on a silicon wafer andannealed in vacuum at 150° C. for 15 h had θ_(w,adv) and θ_(w,rec)values of 95° and 69°, respectively, similar to those for a polystyrenesurface.

Thus, it can be inferred that the surface at equilibrium is covered bythe lower surface-energy PS block at equilibrium, as expected (surfaceenergy of PS is about 39.3 mJ/m² and that of P4VP is 68.2 mJ/m²; seeJiang et al. Polymer 1998, 39, 2615-2620). However, the quaternizedblock copolymer surfaces had lower contact angles (cf. Table 2),indicating the presence of the pyridinium block at the surface.Interestingly, the receding contact angles were lower for thePS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br andPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br surfaces with hydrophobicsemifluorinated side groups, than the non-fluorinatedPS_(11k)P4VP_(21k)H6Br and PS_(62k)P4VP_(66k)H6Br surfaces. One mayinfer that, in contact with water, the surface-concentration of thehydrophilic pyridinium rings is higher in surfaces with a mixture ofF8H6 and H6 alkyl groups. Moreover, the large contact angle hysteresisindicates that these mixed surfaces are mobile, that is, the surfacescan reconstruct to become hydrophilic in the presence of water.

TABLE 2 Advancing and receding water contact angles on spray-coatedsurfaces. Water CA Surface θ_(w, adv) θ_(w, rec) PS_(11k)P4VP_(21k)H6Br73° 16° PS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br 63°  8°PS_(62k)P4VP_(66k)H6Br 55° 16° PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br 56° 7° P(4VP-r-BMA)_(300k)H6Br 99° 10°

Quaternization of the 4-vinylpyridine polymer did not always result inlowering of the contact angles. A spray-coated surface ofpoly(4-vinylpyridine-ran-n-butyl methacrylate), annealed at 60° C. for24 hours, had advancing and receding water contact angles of 72° and20°, respectively. However, the corresponding quaternized polymer,P(4VP-r-BMA)_(300k)H6Br, had θ_(w,adv) and θ_(w,rec) values of 99° and10°, respectively (cf. Table 2). The higher advancing water contactangle is attributed to a layer of hydrophobic n-hexyl chains coveringthe pyridinium rings. The lower surface energy —CH₃ (˜24 mJ/m²) and—CH₂—(˜31 mJ/m²) groups of the alkyl side chains will be preferentiallypresent at the air—polymer interface, covering the higher energypyridinium groups (see Pittman, A. G. In Fluoropolymers, High PolymersSeries XXV; Wall, L. A., Ed;. Wiley-Interscience: New York, 1972. p419-449).

NEXAFS Spectroscopy. NEXAFS spectroscopy allows the determination of therelative numbers of carbon and nitrogen atoms, and also the orientationof bonds in the surface region. The size of the edge jump isproportional to the number of absorbing atoms (C or N), and thus varieswith surface concentration (see Stöhr J. NEXAFS Spectroscopy.Springer-Verlag: New York, 1996; Chapter 7, p 211). The edge jump isgiven by the difference of the electron yield about 30 eV above theionization threshold (320 eV in C 1s NEXAFS spectra and 430 eV in N 1sspectra) and the electron yield just below the first resonance. Asdiscussed above, the latter is approximately 0 for both C 1s and N 1sspectra, and the C 1s edge jump has been set to unity.

The NEXAFS spectra reported here were normalized such that the carbonedge jump was the same (=1) for all the surfaces. Hence, the magnitudeof the nitrogen edge jump is proportional to the surface concentrationof nitrogen atoms relative to carbon. Moreover, a comparison of theintensity of the C 1s→π* peak (near 285.7 eV for P4VP), is anotherindication of the presence or absence of pyridinium groups at thesurface. Using NEXAFS spectroscopy, a comparison of the surfacepyridinium concentrations can be made in a dry state, which thebacterial cells are likely to encounter when they initially contact thesurface. Spectrophotometric titration of surface pyridinium groups,involving immersion of the surfaces in aqueous solution of fluoresceindye, has been described by Tiller et al. (Proc. Natl. Acad. Sci. U.S.A.2001, 98, 5981-5985).

N-hexylpyridinium surfaces. FIG. 4 shows the C 1s and N 1s NEXAFSspectra of surfaces prepared using P4VP_(60k)H6Br polymer as well as theP4VP_(60k) precursor. The asymmetry in the shape of the C₁s→π*peak isattributed to a 1s core level shift arising from differences in thepartial charges on the carbon atoms at ortho and meta positions. Theortho atoms that are bonded directly to the nitrogen are more positivethan the meta atoms which are further away from the nitrogen (seeKolczewski et al. J. Chem. Phys. 2001, 115, 6426-6437). Similarly, thepartial charge on the nitrogen atom will be higher than those on thecarbon atoms of the ring. Hence, the difference in the resonanceenergies for the 1s→π* transition before and after quaternization, ismuch more pronounced in the N 1s spectra than in the C 1s spectra (seeIto et al. J. Am. Chem. Soc. 1997, 119, 6336-6344).

Quaternization had a strong effect on the position of the N 1s→π*resonance, which shifted to a higher energy by about 2 eV (from 400.7 eVfor the un-quaternized P4VP_(60k) polymer, to 402.7 eV for thequaternized P4VP_(60k)H6Br polymer). Similar shifts were observed in theXPS nitrogen signals of quaternized polymers discussed above and havebeen reported for P4VP and protonated P4VP by Fujii et al (J. Am. Chem.Soc. 2005, 127, 16808-16809).

In FIG. 4, it is seen that the intensity of C 1s→π*_(C═C, C═N) peak isnotably lower for the quaternized surface. The N 1s→π* resonance forthis surface (FIG. 4 b) is also lower in intensity compared to theP4VP_(60k) surface. The observed decrease is, in most part, due to thedecrease in the transition probability (the number of electrons excitedper unit time from the 1s shell) after quaternization; and also in thecase of the N 1s resonances due to the fact that the nitrogen to carbonatomic ratio in the polymer decreases from 1/7 to 1/13 afterquaternization. If the surface composition is uniform (same as that inthe bulk), the relative intensities of the N 1s→π* peaks at 400.7 eV inthe NEXAFS spectra of the un-quaternized and quaternized polymers is anindication of the degree of quaternization. The nitrogen edge jumps ofthe spectra in FIG. 4 b were normalized to the same value so that thecomparison was made for the same number of nitrogen atoms in both thesurfaces. From the decrease in the intensity of the π* resonance at400.7 eV in the normalized spectra, it was inferred that more than 90%of the pyridine groups have undergone the quaternization reaction.

The degree of quaternization estimated using this procedure will differfrom that obtained by more conventional methods (such as IR or elementalanalysis) if the low surface-energy alkyl groups in the quaternizedpolymer form a thin layer at the surface covering the higher surfaceenergy pyridinium rings. The maximum thickness of this layer can beestimated to be about 7.7 Å, corresponding to a fully stretched n-hexylchain attached to the pyridinium nitrogen. In such a case, the degree ofquaternization obtained from the NEXAFS spectra can be a slightover-estimate.

Rather different results were obtained from thepolystyrene-block-poly-(4-vinylpyridine) surfaces. Quaternization of thePS-b-P4VP polymer resulted in an increase in the intensity of the N 1sresonances in the NEXAFS spectra. The C 1s NEXAFS spectra of thePS_(11k)P4VP_(21k) and PS_(62k)P4VP_(66k) surfaces in FIG. 5 a areindistinguishable from the spectrum of PS homopolymer. Thus, theun-quaternized block copolymer surfaces are almost completely covered bythe lower surface-energy PS block, which fully supports theinterpretation of the contact angle results.

Using PS-b-P4VP block copolymers in which the P4VP block wasend-functionalized with 3,3,3-trifluoropropyldimethylchlorosilane, Jianget al. found that the P4VP block segregated to the surface because ofthe low surface-energy —CF₃ group at its end (Polymer 1998, 39,2615-2620). However, the PS_(11k)P4VP_(21k) and PS_(62k)P4VP_(66k) blockcopolymers used in our study did not show surface segregation of thehigher surface-energy block, even though the P4VP blocks were terminatedwith perfluorooctyl groups. The reason for the difference probably liesin the fact that the block copolymer studied by Jiang and coworkers hada relatively low molecular weight (14 kDa total) compared to those usedin the present study. A single perfluorooctyl group at the end of ourlonger P4VP blocks was unable to bring these to the surface.

To displace the PS block from the surface, the decrease in P4VP blocksurface energy contributed by the perfluorooctyl groups must compensatefor the increased energy of the exposed P4VP surface. This compensationwill require a high areal density of perfluorooctyl groups resulting inthe necessity of the P4VP chains to stretch away from the surface. Thefree energy penalty for the required P4VP stretching increases as theP4VP block length increases and, thus, above some block length, theperfluorooctyl end group will be ineffective in bringing the P4VP blockto the surface.

While the PS_(62k)P4VP_(66k) block copolymer surface did not show any N1s signal, the 4-vinylpyridine block could be detected in the N 1sNEXAFS spectrum of the PS_(11k)P4VP_(21k) polymer. However, theseresonances were much weaker in intensity compared to the P4VPhomopolymer (cf. FIG. 4 b). The radius of gyration of PS with amolecular weight of 62 kDa can be estimated to be about 7 nm (see Cottonet al. Macromolecules 1974, 7, 863-872). The thickness of the PS layercovering the 4VP block is expected to be at least 7 nm, which is largecompared to the expected escape depth of the N 1s Auger electrons fromthe surface. Hence, if the P4VP block is buried below a layer of PSchains, it would not be detected, as observed experimentally for thePS_(62k)P4VP_(66k) surface.

The radius of gyration of PS with a molecular weight of 11 kDa is about2.9 nm, comparable to the escape depth of the N 1s Auger electrons.Thus, some detection of the Auger electrons resulting from N 1stransitions would be expected for the PS_(11k)P4VP_(21k) surface, asseen experimentally.

When the PS-b-P4VP polymer is quaternized with 1-bromohexane, the lowersurface energy —CH₃ and —CH₂— groups of the alkyl side chains would bethermodynamically favored at the air-polymer interface over the phenylrings of the PS block. In contrast to the un-quaternized polymers, thepresence of the pyridinium block at the surface is evident from theNEXAFS spectra of FIG. 5 (c and d). The N 1s resonances are higher inintensity, compared to the spectra in FIG. 5 b, especially in the caseof the PS_(62k)P4VP_(66k)H6Br surface. The spectra of the blockcopolymers with different molecular weights, PS_(11k)P4VP_(21k)H6Br andPS_(62k)P4VP_(66k)H6Br, now become almost identical. The N 1s resonancesin the NEXAFS spectra of the quaternized diblock copolymers were,however, lower in intensity than those for the homopolymerP4VP_(60k)H6Br due to the presence of some phenyl rings at the surface(cf. FIG. 4 b and FIG. 5 d). The effect of PS block at the surface isalso evident in the higher intensity of the C 1s→π* resonance intensitycompared to the P4VP_(60k)H6Br homopolymer (cf. FIG. 4 a and FIG. 5 c).

NEXAFS analysis of the surfaces used for bacterial tests. The C 1s and N1s NEXAFS spectra of the surfaces used in the bacterial assays are shownin FIG. 6 and FIG. 7. These surfaces were prepared by spray-coating thequaternized polymers on SEBS covered glass microscope slides, aspreviously discussed. FIG. 6 compares the NEXAFS spectra of thefluorinated and non-fluorinated pyridinium diblock copolymers,PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br and PS_(62k)P4VP_(66k)H6Br,respectively. The differences in the NEXAFS spectra of thePS_(62k)P4VP_(66k)H6Br surfaces prepared by the spin-coating (FIG. 5)and the spray-coating (FIG. 6) techniques are attributed to thedifferent processing conditions used for the two surfaces. Thespin-coated samples were annealed at 150° C., which is above the glasstransition temperature of the polystyrene block, while the spray-coatedsamples were dried at 60° C.

The is 1s→σ*_(C—F) resonance in the C 1s spectrum ofPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br, near 293 eV in FIG. 6 a, showed thepresence of the semifluorinated alkyl group, and hence the pyridiniumblock, at the surface. The intensity of the σ*_(C—F) resonance wasindependent of the X-ray incident angle. Hence, the semifluorinated sidegroups were not oriented the surface. The N 1s resonances, and also theedge jump, were higher in intensity for the fluorinated pyridinium blockcopolymer surface than the corresponding non-fluorinated polymer (cf.FIG. 6 b), indicating a higher surface concentration of pyridinium ringsin the former surface.

To investigate the effect of molecular weights of the PS and P4VP blockson the surface composition of the quaternized polymers, the intensitiesof the carbon and nitrogen K-edge resonances were compared. For both thenon-fluorinated and fluorinated PS_(11k)P4VP_(21k) andPS_(62k)P4VP_(66k) pyridinium block copolymers, (i) the intensity of theC 1s→π* transition was higher in the case of the higher molecular weightpolymers, and (ii) the edge jump and the N edge resonances were lower inintensity for the higher molecular weight polymers. These observationsindicate that the surface concentration of polystyrene units was higherin the case of the PS_(62k)P4VP_(66k) polymers than thePS_(11k)P4VP_(21k) polymers. Also, as seen from FIG. 6 b, the surfaceconcentration of N⁺ atoms is higher for the fluorinatedPS_(61k)P4VP_(66k) surface.

FIG. 7 compares the NEXAFS spectra of the P(4VP-r-BMA)_(300k) andP(4VP-r-BMA)_(300k)H6Br surfaces. The absence of the π* peakcorresponding to un-quaternized pyridine rings in the N 1s spectrum,reflects the almost complete quaternization of the precursor polymer,which is in accord with the results from IR spectroscopy.

X-ray Photoelectron Spectroscopy of Surfaces used for AntibacterialTests

The relative numbers of carbon and nitrogen atoms at the surfaces of thepyridinium polymers used in the antibacterial assays were also comparedusing XPS. Bilayer coatings were prepared by spray coating the polymerson SEBS covered glass slides, followed by drying at 60° C. in vacuum toremove solvent. All XPS data were collected with a 0° electron emissionangle (along the surface normal). FIG. 8 shows the N 1s XPS spectra forthe P4VP homopolymer before and after quaternization with 1-bromohexane.Upon quaternization, the nitrogen peak shifted to a higher bindingenergy. The small peak at 399 eV is due to the nitrogen atoms that hadnot undergone the quaternization reaction. By comparing the areas underthe two peaks, the percentage of nitrogen atoms that were quaternizedcould be calculated. As seen in Table 3, all the pyridinium polymersshowed a high degree of quaternization.

TABLE 3 Percent of pyridinium groups quaternized. Polymer %Quaternization P4VP_(60k) 0 P4VP_(60k)H6Br 95.2% PS_(11k)P4VP_(21k)H6Br95.5% PS_(62k)P4VP_(66k)H6Br 90.9% PS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br93.1% PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br 94.3%

The C 1s XPS spectra of the fluorinated pyridinium block copolymers(FIG. 9 a) showed distinct —CF₂— and —CF₃ peaks at binding energies of292 eV and 294 eV, respectively. Although a small number of C—F carbonatoms from the perfluorooctyl end groups of the PS-b-P4VP precursors(cf. block copolymer 1 in Scheme 1) are expected to be present in theotherwise non-fluorinated PS_(11k)P4VP_(21k)H6Br and PS_(62k)P4VP H6Brsurfaces, the low intensity peaks seen near 292 eV in FIG. 9 b are theshake-up peaks. The shoulder at 286 eV is characteristic of carbon atomsbonded to nitrogen atoms (see Cen et al. Langmuir 2003, 19,10295-10303).

The areas under the C—N peaks were lower for the non-fluorinated blockcopolymers than the fluorinated polymers, suggesting a higherconcentration of quaternary nitrogen in the fluorinated surfaces.Moreover, the areas of the N 1s peaks near 402 eV (FIG. 9 c and FIG. 9d) were correspondingly lower for the non-fluorinated polymers. Thus,the lower molecular weight polymers had more N⁺ atoms at the surfacethan their higher molecular weight counterparts, and the fluorinatedpolymers had more quaternized nitrogen at the surface than thenon-fluorinated pyridinium block copolymers.

The results of NEXAFS spectroscopy and XPS may be summarized as follows.Quaternization of PS-b-P4VP with 1-bromohexane resulted in the presenceof the higher surface energy P4VP block at the surface, which wasotherwise buried below the PS block. The relative number of N⁺ atoms atthe surface was further enhanced when 6-perfluorooctyl-1-bromohexane wasused. Partial quaternization of PS-b-P4VP with F8H6Br resulted in ahigher surface concentration of N⁺ compared to block copolymersalkylated using H6Br alone. The PS_(62k)P4VP_(66k) block copolymers withhigher weight fractions of PS, showed higher surface concentrations ofPS.

Antibacterial Assay. The antibacterial activity of the pyridiniumsurfaces were evaluated by performing a viable count on S. aureus cellssprayed onto the surfaces. As seen in FIG. 10A, a large number ofbacterial colonies formed on the untreated glass slide, which is notexpected to have any bactericidal activity.

Assuming that the same number of S. aureus cells were sprayed onto theglass control and test surfaces, the relative number of colonies on theglass and test surfaces represents the fraction of the sprayed cellsthat remained viable on the test surfaces. The viable counts were 15 to30% lower on the PS_(11k)P4VP_(21k)H6Br (FIG. 10B) andPS_(62k)P4VP_(66k)H6Br (FIG. 10C) surfaces compared to the glasscontrol. While the non-fluorinated diblock copolymers had a large numberof bacterial colonies, only somewhat lower than that on uncoated glass,the fluorinated pyridinium polymers (FIG. 10E and FIG. 10F) showed analmost 100% decrease in the viable count. The lengths of the pyridiniumblocks in both the sets of polymers were the same, but quaternizationwith F8H6Br caused a significant increase in the bactericidal activityof the surfaces.

The enhanced activity is attributed to the differences in surfacecompositions and molecular organizations. Both NEXAFS spectroscopy andXPS showed that the surface concentration of the pyridinium rings, andhence the surface charge density, was higher in the case of thefluorinated polymers, which is also consistent with the lower watercontact angles observed for these surfaces. A higher charge density isexpected to result in stronger electrostatic interactions between thecells and the surface, which in turn would lead to cell death by variousmechanisms. The concentration of pyridinium rings, however, cannot bethe only factor affecting antibacterial activity. The surfaceconcentration of the quaternary nitrogen was higher forPS_(11k)P4VP_(21k)H6Br than PS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br. However,the bactericidal effect of the non-fluorinated surface was significantlylower. It is not known conclusively whether the rigid and highlyhydrophobic perfluoroalkyl helices have a greater ability to disrupt thebacterial cell membrane. Nevertheless, the non-polar nature and arod-like conformation of the fluoroalkyl helices could be responsiblefor the higher antibacterial activity of the fluorinated surfaces.

The viable count for the relatively high molecular weightP(4VP-r-BMA)_(300k)H6Br polymer (FIG. 10D) was 60±12% lower than that onglass. Despite its high molecular weight, its bactericidal efficiencywas less than that of the PS_(11k)P4VP_(21k)(F8H6_(0.3)H6)Br andPS_(62k)P4VP_(66k)(F8H6_(0.3)H6)Br surfaces. This suggests that inaddition to molecular weight, the molecular organization at the surfaceplayed a crucial role in antibacterial activity. The reason for thereduced activity was partially evident from the contact anglemeasurements. The advancing water contact angle on the spray-coatedsurface of the P(4VP-r-BMA)_(300k)H6Br polymer was about 99°. Such ahigh angle is indicative of a very dense layer of alkyl groups coveringthe pyridinium rings, which is believed to be unfavorable forbactericidal activity.

The S. aureus cells used in the antibacterial assays were in theexponential phase of growth and capable of cell division. The lowernumber of bacterial colonies on the pyridinium surfaces could be eitherthrough interference with cell division, or by causing majordisorganization of the cell membrane resulting in cell death. TheBacLight staining method confirmed that the test surfaces causeddisruption of the cell membrane within 15 min of contact. BacLightemploys two nucleic acid stains: the green-fluorescent SYTO® 9, whichhas excitation and emission maxima at 480 nm and 500 nm, respectively,and the red-fluorescent propidium iodide (PI), which has excitation andemission maxima at 537 nm and 620 nm, respectively. See Biggerstaff etal. Molecular and Cellular Probes 2006, 20, 141-146; and Boulos et al.J. Microbiol. Meth. 1999, 37, 77-86.

Besides their spectral characteristics, SYTO® 9 and PI have differentabilities to penetrate bacterial cell membranes and different bindingaffinities toward nucleic acids. SYTO® 9 can freely permeate intact cellmembranes. It is essentially non-fluorescent in the free-state, but itsfluorescence quantum yield increases by 1000-fold or more upon bindingto nucleic acids. In contrast, PI penetrates only cells with damagedmembranes. Propidium iodide has a higher affinity toward nucleic acids,displaces the less strongly bound SYTO® 9 thereby reducing the intensityof the green fluorescence, and itself fluoresces red. The fluorescencequantum yield of PI increases 20-30 fold upon binding to nucleic acids.Thus, by the suppression of the intensity of green fluorescence and anenhancement in the red fluorescence, bacteria with damaged membranesappear red while those with intact membranes appear green.

S. aureus cells on an uncoated glass slide and on a glass slide coatedwith the P(4VP-r-BMA)_(300k)H6Br polymer were stained. Cells with intactcell membranes were stained green and those with damaged membranes arestained red. Almost all of the cells on the glass control were stainedgreen, indicating intact and possibly viable cells. The cells on thesurface of the quaternized polymer were stained red, suggestingdisruption of the cell membrane. Thus, the antibacterial activity ofpyridinium surfaces seems to be through the loss of membrane integrity,rather than inhibition of cell division.

Conclusions. Pyridinium block copolymers with fluorinated side chainswere synthesized by quaternization reaction of a semifluorinated alkylbromide with polystyrene-block-poly(4-vinylpyridine). Surfaces of thefluorinated block copolymers were found to be more effective indecreasing the viability of airborne Staphylococcus aureus than N-hexylpyridinium surfaces. NEXAFS and contact angle measurements showed thatfluorination resulted in an increase in the number of pyridinium ringsat the surface which, in general, correlated with a higher antibacterialactivity. In addition, the fluoroalkyl side chains may be intrinsicallyfavorable for disruption of the bacterial cell membrane due to theirrigidity and hydrophobicity.

Surfaces with a dense layer of alkyl side chains covering the pyridiniumgroups showed higher water contact angles, lower N 1s signals in NEXAFSspectroscopy, and were found to exhibit lower antibacterial activity. Incontrast, when alkyl (or fluoroalkyl) side groups were not denselypacked and the surface concentration of the pyridinium nitrogen wassufficiently high, a greater antibacterial activity was observed.Molecular weight does not seem to be a limiting factor in determiningantibacterial activity. The fluorinated pyridinium block copolymer witha relatively low P4VP block molecular weight of 21 kDa (degree ofpolymerization ˜200), showed almost 100% bactericidal effect. Thepyridinium surfaces were also found to inhibit the growth of spores ofthe marine alga Ulva linza when immersed in seawater (see Krishnan etal. Polymer Preprints (American Chemical Society, Division of PolymerChemistry) 2005, 46, 1248-1249).

Example 3 Interaction of Ulva and Navicula Marine Algae with Surfaces ofPyridinium Polymers with Fluorinated Side-Chains

Introduction: The formation of a microbial biofilm of bacterial cells,algal spores, diatoms and protozoa is an important initial step in themarine biofouling process. Surfaces prepared using quaternized4-vinylpyridine polymers are antibacterial and can prevent the formationof bacterial biofilms. The antibacterial activity of these polymers isbelieved to arise from the cationic binding of the cell membrane,resulting in a disruption of the membrane and subsequent leakage of thecytoplasmic materials. Ulva zoospores and Navicula diatoms aremicroalgae commonly encountered in biofilms that develop on ship-hulls.Both are unicellular organisms and attach to surfaces throughextracellular polymeric substances (EPS) that are adhesive in nature.

Settlement of a free-swimming Ulva zoospore on a surface is influencedby physico-chemical properties of the surface (such as chemicalcomposition and wettability) and surface topography. Adhesion isachieved by the secretion of a hydrophilic glycoprotein that crosslinksrapidly after release from the spore.

The work reported in this Example investigated whether 4-vinylpyridiniumpolymers that formed bactericidal surfaces could also function as marineantifouling surfaces. Polystyrene-block-poly(4-vinylpyridine) (PS/P4VP)was quaternized with alkyl and fluoroalkyl bromides, andsurface-activity against Ulva and Navicula was studied. The number ofUlva zoospores and Navicula cells settling and attaching to the thesesurfaces was determined and compared to hydrophobic poly(dimethylsiloxane) and hydrophilic glass controls. A block copolymer withfluoroalkyl side chains, but without the positive charge of the4-vinylpyridinium polymers was also studied. The adhesion strengths ofalgal cells were characterized by the degree of removal under watershear stress in a turbulent flow channel.

Experimental. Scheme 5 shows three types of surface-active blockcopolymers that were studied. Polystyrene-block-poly(4-vinylpyridine)(PS/P4VP) block copolymers were synthesized by anionic polymerization(Krishnan et al. Polym. Mater. Sci. Eng. 2004, 91, 814). Polymers withPS and P4VP block molecular weights of 11 kDa/21 kDa and 62 kDa/66 kDawere quaternized with ω-6-perfluorooctyl-bromohexane and 1-bromohexaneto obtain block copolymers with semifluorinated side chains (I).

Synthesis of semifluorinated alkyl bromide. Three grams (5.77 mmol) ofω-6-perfluorooctyl-hexanol and 3 g (9.05 mmol) of CBr₄ were dissolved ina mixture of 6 mL anhydrous THF and 12 mL anhydrous methylene chlorideand cooled to −5° C. Triphenylphosphine (2.37 g, 9.05 mmol) was thenadded in small portions over a period of 15 minutes. After stirring for1 hour at −5° C. and 6 hours at room temperature, solvent was evaporatedfrom the reaction mixture under vacuum, and ca. 50 mL diethyl ether wasadded. The insoluble solid (triphenylphosphineoxide byproduct) wasseparated by filtration, and the filtrate concentrated to obtain thecrude product that was purified by passing through a short silica gelcolumn with diethyl ether as the elution solvent.

Quaternization using ω-6-perfluorooctyl-bromohexane. One gram (4.92 mmol4-VP) of the PS/P4VP block copolymer (62 k/66 k) and 0.8630 g (1.48mmol) of ω-6-perfluorooctyl-bromohexane were dissolved in 10 mL of DMFand heated to 80° C. for ca. 24 hours. 1-Bromohexane (5.88 g, 35.6 mmol)was added and the reaction was continued further for 24 hours at 80° C.After cooling to room temperature the polymer was precipitated indiethyl ether at 0° C. PS/P4VP(62/66)F8H6Br-30% was prepared by reacting30 mol % of F(CF₂)₈(CH₂)₆Br (based on 4-VP) with the 62 k/66 k PS/P4VPblock copolymer. PS/P4VP(11/21)F8H6Br-30% and PS/P4VP(62/66)F8H6Br-50%block copolymers were similarly prepared. A random copolymer of 4-VP andn-butylmethacrylate copolymer with a molecular weight of 300,000 g/moland 10 wt % BMA were also quaternized with 1-bromohexane (II).

Side-chain liquid crystalline block copolymers with semifluorinatedgroups (III) were prepared by polymer analogous reactions onpolystyrene-block-poly(isoprene) (PS/PI) copolymer with block molecularweights of 10 kDa/12 kDa. The procedure described Wang and co-workers(Macromolecules 1997, 30, 1906) was used to prepare thePS/PI(10/12)F10H9 block copolymer. The block copolymers werecharacterized by IR spectroscopy on a KBr salt plate.

Preparation of test surfaces. Bilayer Coatings were Prepared bySpray-Coating the block copolymer solutions on a glass microscope slidecoated with a PS-block-poly(ethylene-ran-butylene)-block-PSthermoplastic elastomer (SEBS, Kraton G1652). The PS blocks in thebottom (SEBS) and top (SABC) layers are expected to mix at theinterface, welding the two layers. 1.5% (w/v) solutions of thequaternized polymers in chloroform (or chloroform-methanol mixture), and2% (w/v) solution of (III) and SEBS (9:1 mass ratio) inα,α,α-trifluorotoluene and toluene (2:1 volume ratio) were used forspray-coating. The former surfaces were dried in vacuum at 60° C., andthe PS/PI(10/12)F10H9 surfaces annealed at 120° C. for 24 hours.

Algal Assays. Fertile plants of Ulva linza were collected from Wemburybeach, UK (50° 18′N; 4° 02′W). Zoospores were released and treated asdescribed by Wang and co-workers (ibid.). Each slide was exposed to 10mL of a 1.5×10⁶ spore/mL suspension in seawater for 1 hour. Settledzoospores were counted. Adhered zoospores were cultured for 10 days toproduce sporelings (young plants), and the resulting biomass wasquantified by measurement of the chlorophyll a content. To measure theattachment strength of the sporelings, biomass was determined before andafter applying a wall shear-stress of 53 Pa in a flow channel. Assayswith diatoms (Navicula) were set up as described by Holland andco-workers Biofouling 2004, 20, 323) Attachment strength was measured ina flow channel and percentage removal calculated as above.

Results and Discussion:

Settlement, Growth and Release of Ulva and Navicula. FIG. 11 shows thesettlement and growth of Ulva zoospores on the pyridinium and isoprenebased block copolymers with semifluorinated side chains. Althoughsettlement was significantly higher on the pyridinium surface comparedto PDMS or glass (cf. FIG. 11 a), the growth of sporelings was lower(cf. FIG. 11 b). Considering the fact that the PS/P4VP(11/21)F8H6Br-30%surface was hydrophilic (advancing and receding water contact angles ofca. 56° and 7°, respectively), high settlement on this surface isopposite of what is expected from previous results on the influence ofsurface wettability on settlement of Ulva. Using alkane thiolsterminated with methyl and hydroxyl groups, and mixtures of the two,Finlay et al. found that settlement of Ulva was greatest on ahydrophobic surface (Finlay et al. Integr. Comp. Biol. 2002, 42, 1116).

An important difference between the CH₃ and OH self-assembled monolayersand the pyridinium surface is that the latter is charged. Favorableelectrostatic interactions between the negatively charged spores (or theEPS) and the polymer surface could have promoted spore-settlement, buttheir growth was inhibited, possibly due to the antimicrobial activityof the surface. The non-polar PS/PI(10/12)F10H9 surface with advancingand receding water contact angles of 123° and 85°, respectively, on theother hand, showed lowest settlement and growth, and a relatively highbiomass-removal of ca. 70% (compared to ca. 24% from the pyridiniumsurface and ca. 11% from glass).

From a study using PEGylated and fluorinated (III) block-copolymersurfaces, we found that the adhesion strength of Navicula diatoms waslowest on a hydrophilic surface, which concurs with previousobservations (see Holland et al. Biofouling 2004, 20, 323). However, asseen from FIG. 12, the adhesion to some of the hydrophilic pyridiniumsurfaces was stronger in comparison to the hydrophobicPS/PI(10/12)F10H9. This could again be attributed to electrostaticinteractions, which are absent in fluorinated and PEGylated surfaces.

Conclusions. The settlement of Ulva zoospores on thePS/PVP(11/21)F8H6Br-30% surface was unexpectedly high, possibly due toelectrostatic interactions between the spores and the surface. However,this surface inhibited the growth of spores, and the amount of biomassafter a 10-day growth period was lower than that on PDMS or glass.Release of settled Navicula diatoms was easier from the relatively highmolecular weight P(4VP-co-BMA)H6Br and the PS/PI(10/12)F10H9 surfacescompared to PDMS. The pyridinium surfaces are expected to show astronger antimicrobial effect against Ulva compared to Navicula whosecells are surrounded by a protective siliceous cell wall (frustules).

Example 4 Pyridinium Polymers with PEGylated and Semifluorinated SideGroups

Pyridinium block copolymers with ω-6-perfluorooctyl-bromohexane and1-bromohexane side chains have shown promising antibacterial activityagainst the air-borne pathogen, S. aureus. They have also shownantimicrobial activity against Ulva zoospores, inhibiting the growth ofsporelings. Scheme 6 shows the structure of the amphiphilic pyridiniumblock copolymer with PEG and fluoroalkyl side chains. The pyridiniumblock copolymer is illustrated with mixed semifluorinated and PEGylatedside groups. A PS_(62k)P4VP_(66k) diblock copolymer was used. About 30%of the pyridine groups were quaternized with semifluorinated alkylbromide and the remaining with PEG bromide.

The amphiphilic character was evident from the water contact anglemeasurements, which were highly dependent on the temperature at whichthe surfaces were annealed (Table 4). A 3% (w/v) solution of the blockcopolymer in chloroform was used, and the surfaces were annealed at 120°C. or 60° C. in vacuum for 12 hours.

TABLE 4 Advancing and receding water contact angles Anneal temp. θ_(A)θ_(R) Spin-coated 120° C.  102°  9° Spin-coated 60° C. 71° 11° Spray-coated 60° C. 91° 9°

The relatively high advancing angle and the low receding angle suggestthe presence of the pyridinium block at the surface. The low recedingwater contact angle is due to the ionic pyridinium groups and thehydrophilic PEG chains. XPS spectra of the spray-coated surface areshown in FIG. 13. The high resolution C1s spectra were obtained atelectron emission angles of 0° and 75° with respect to surface normal. Ahigher surface concentration of CF₂ and CF₃ groups is evident from the75° emission angle spectrum, which probes thinner surface layers than 0°emission.

The pyridinium surfaces were found to be water sensitive due to a highcontent of hydrophilic PEG groups, in addition to the ionic nature ofthe pyridinium rings. The polymer shown in Scheme 6 does not haven-hexyl side chains. Current understanding of antibacterial activity ofpyridinium polymers is that the groups attached to the pyridinium ringshould have an optimal hydrophilic-lipophilic balance to penetrate thelipid bilayer of bacterial cell membrane. A higher fluoroalkyl contentshould improve water resistance, and antifouling performance.

Example 5 Free Radical Polymerization Methods

Anionic polymerization to synthesizepolystyrene-block-poly(4-vinylpyridine) block copolymers was discussedabove. The anionic mechanism allowed relatively rapid polymerization andfacile end-functionalization of the 4VP block with(heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane. However,the polymerization reaction is highly air- and moisture-sensitive andrather stringent purity of chemicals is required. Controlled freeradical polymerization, possibly more versatile than anionicpolymerization, can also be used to prepared the polymers of theinvention, including those containing 4-vinylpyridine groups.

Polystyrene-block-poly(4-vinylpyridine-co-styrene) was prepared bynitroxide mediated controlled free radical polymerization. Benzoylperoxide and TEMPO were used as the initiator and capping agent(stabilizer), respectively, in the synthesis of the PS macroinitiator(Scheme 7). The PS block mol. wt. was about 20,000 g/mol. The PSmacroinitiator was then used to copolymerize styrene and 4-vinylpyridineto obtain polystyrene-block-poly(4-vinylpyridine-ran-styrene) diblockcopolymer. The P(4VP-r-S) block had a molecular weight of about 70,000g/mol, and consisted of about 30 mol % styrene and 70 mol %4-vinylpyridine. The pyridine groups were quaternized with1-bromohexane.

Surfaces prepared from solutions of this polymer in chloroform orchloroform-methanol mixture showed good antibacterial activity againstS. aureus. Moreover, spray-coated surfaces of this polymer did not cloudupon exposure to water, and hence can also be used as a marineanti-bactericidal coating.

FIG. 14 compares the NEXAFS spectra of spray-coated bilayer coatings ofdifferent pyridinium polymers on SEBS substrates. Of these, the polymerPS_(20k)P(4VP_(0.7)-r-S_(0.3))_(70k)H6Br was obtained from blockcopolymer precursor synthesized using nitroxide mediated polymerization.The N 1s resonances were higher in intensity in the case of thePS_(20k)P(4VP_(0.7)-r-S_(0.3))_(70k)H6Br compared to the fluorinated andnon-fluorinated PS_(62k)P4VP_(66k) pyridinium surfaces. Thus, thesurface concentration of pyridinium rings is higher forPS_(20k)P(4VP_(0.7)-r-S_(0.3))_(70k)H6Br, which is expected to impartgood antibacterial activity.

Controlled free radical polymerization of 4-vinylpyridine can be carriedout using any of the several nitroxide initiator systems reported in theliterature, for example,N-tert-butyl-N-(1-diethylphosphono-2,2,-dimethyl)propyl nitroxide(DEPN, 1) with 2,2′-azobisisobutyrinitrile (AIBN), or the unimolecularinitiators (2) and (3) (Scheme 8). See Benoit et al. J. Am. Chem. Soc.2000, 122, 5929-5939; and Hawker et al. Chem. Rev. 2001, 101, 3661-3688,respectively for DEPN and unimolecular inhibitors, respectively. Lee etal. have recently reported a reversible addition-fragmentation chaintransfer (RAFT) method to preparepolystyrene-block-poly(4-vinylpyridine). See Polymer 2006, 47,3838-3844.

End-functionalization with semifluorinated groups can be achieved bygrowing a short third block of fluorinated monomer as shown in Scheme 9,where Rf is a semifluorinated alkyl group.

The structure shown in Scheme 9 would be obtained if the unimolecularinitiator 3 (Scheme 8) were used in the triblock copolymer synthesis.

Example 6 Marine Antifouling Characteristics of Poly(N-hexylpyridiniumbromide-ran-n-butyl methacrylate), P(4VP-r-BMA)_(300k)H6Br, EvaluatedUsing the Green Alga, Ulva

Polymer synthesis. Six grams of poly(4-vinylpyridine-ran-butylmethacrylate), with a molecular weight of 300 kDa and 10 wt % of n-butylmethacrylate, was reacted with 8 mL of 1-bromohexane in 60 mL ofnitromethane at 80° C. for 2 days followed by precipitation of thepolymer in diethyl ether, as illustrated in Scheme 10.

Preparation of test surfaces. Surfaces were prepared on 3 inch×1 inchglass microscope slides. To improve adhesion of the pyridinium polymerto glass,polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (KratonSEBS G1652) was first spin-coated on the glass slides using a 10% (w/v)solution in toluene and annealed in a vacuum oven at 120° C. for 12hours. A 1.5% (w/v) solution of P(4VP-r-BMA)_(300k)H6Br was then sprayedon the SEBS-coated glass slides (heated to 80° C. on a hot-plate) usinga Badger Model 250 airbrush (50 psi nitrogen gas pressure). About 3 mgof the pyridinium polymer was used per cm² of the surface. Thespray-coated surfaces were dried in a vacuum oven at 60° C. for 24hours. Surfaces of unquaternized random copolymer P(4VP-r-BMA)_(300k)were similarly prepared to be used as controls.

Ulva assays. Slides were leached for 6 days in circulating de-ionizedwater and immersed in seawater for one hour before the start of theexperiment. Fertile plants of Ulva linza were collected from WemburyBeach, England (50° 18′ N; 4° 02′ W). Zoospores were released andprepared for attachment experiments as described by Callow et al. (J.Phycol. 1997, 33, 938-947). Slides (3 replicates) were settled withspores using the same stock and concentration of spores that were usedfor the sporeling studies (6 replicates). Ten-milliliter of zoosporesuspensions were pipetted into individual compartments of polystyreneculture dishes (Fisher), each containing a glass microscope slide. Thedishes were incubated in the dark at about 20° C. After 1 hour theslides were gently washed in seawater to remove zoospores that had notattached. The density of zoospores attached to the surface was countedon each of 3 replicate slides using an image analysis system attached toa fluorescent microscope. Spores were visualized by autofluorescence ofchlorophyll. Counts were made for 30 fields of view (each 0.17 mm²) oneach slide.

Growth of sporelings. Spores were allowed to settle for 1 hour indarkness. After washing, sporelings were cultured in enriched seawatermedium in individual (10 mL) wells in polystyrene dishes underilluminated conditions. The medium was refreshed every 2 days and thesporelings cultured for 7 days. Strength of attachment of sporelings wasassessed by exposing single slides of each treatment to the water jet ata range of water pressures. Sporeling biomass was determined in situ bymeasuring the fluorescence of the chlorophyll contained within thesporelings that covered the slides, in a Tecan fluorescent plate reader.Using this method the biomass was quantified in terms of relativefluorescent units (RFU). The RFU value for each slide is the mean of 70point fluorescence readings. Acid washed glass slides and PDMS surfaces(T2 Silastic) were also included as standards.

Navicula assays. Navicula cells were cultured in F/2 medium contained in250 ml conical flasks. After 3 days the cells were in log phase growth.Cells were washed 3 times in fresh medium before harvesting and dilutingto give a suspension with a chlorophyll a content of approximately 0.3μg/mL. Cells were settled in individual dishes containing 10 mL ofsuspension in natural daylight at ˜20° C. After 2 hours the slides werevery gently washed in seawater to remove cells which had not properlyattached. The density of cells attached to the surface was counted oneach slide using an image analysis system attached to a fluorescentmicroscope. Counts were made for 30 fields of view (each 0.064 mm²) oneach slide.

Strength of attachment. Slides settled with Navicula were exposed toshear stresses in a water channel. Glass and PDMS standards wereincluded. The number of cells remaining attached was compared withunexposed control slides (used to determine attachment as above). Onmost surfaces the cells were counted using the image analysis system asdescribed above.

Toxicity. An additional slide of each treatment was inoculated withcells of Navicula and grown for 3 days in an illuminated cabinet.Observations were made at 24 hour intervals.

Results and Discussion: Ulva assays. The coatings were not affectedafter a six day immersion in water and remained firmly adhered to glasssubstrates. FIG. 15 shows the spore settlement density on glass, PDMS,the unquaternized P(4VP-r-BMA)_(300k), and the quaternizedP(4VP-r-BMA)_(300k)H6Br surfaces. The settlement density was similar onglass, PDMS and P(4VP-r-BMA)_(300k), but was considerably higher onP(4VP-r-BMA)_(300k)H6Br. The higher settlement is possibly due toelectrostatic interactions between the settling organism and thecationic polymer. Although spore settlement was the highest onP(4VP-r-BMA)_(300k)H6Br, the spores appeared damaged or moribund onthese surfaces.

FIG. 16 shows the images of spores and 7 day old sporelings on glass,PDMS and P(4VP-r-BMA)_(300k)H6Br. The pyridinium surfaces showed poorsporeling growth (FIG. 16 f). Most surfaces showed increased sporelingremoval with increasing water pressure (FIG. 17). Removal fromP(4VP-r-BMA)_(300k)H6Br was distinctly better than from glass or theunquaternized P(4VP-r-BMA)_(300k) surfaces.

Navicula assays. The density of attached diatoms after gentle washingwas similar on all surfaces (FIG. 18). Removal fromP(4VP-r-BMA)_(300k)H6Br was moderate, and higher compared to PDMS (FIG.19). Examination 3 days post-inoculation showed a similar amount ofgrowth on all surfaces. There was no difference in growth between thepyridinium surface and controls indicating that it was not toxic orleaching toxic materials. Observations at 24 hours, when cells were inlog phase growth, showed that cells were motile on all surfaces.

Example 7 Representative Mers of the Polymers

FIG. 20 illustrates various mers of various embodiments of the inventionwherein the mers can be selected in any combination in any order toprepare an antifouling polymer. The polymer shown in FIG. 20 merelyillustrates the variety of mers that can be used in various embodimentsand is not an actual polymer that has been prepared.

The group R2 that is a substituent of the quaternized nitrogen can beany of, for example, alkyl, perfluoroalkyl, semifluorinated alkyl,polyethylene glycol (PEG), or a PEGylated fluoroalkyl group. Thevariable R3 can be any suitable end-functional group, for example,hydrogen, a TEMPO derivative, or a substituted silyl group, such astrimethylsilyl or a dialkyl-partially fluorinated alkyl-silyl group, orother groups as discussed herein.

The specific lengths of alkyl, fluoroalkyl, and ethylene glycol groupsshown in FIG. 20 are representative only and other chain lengths can beemployed. The length of each mer substituent and/or quaternization groupcan be selected in order to achieve the desired physical properties ofthe antifouling coating being prepared.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A polymer comprising a plurality of two-carbon repeating units in apolymer chain, wherein one or more of the two-carbon repeating units ofthe polymer chain have pyridine-containing substituents; and at leastabout 10% of the nitrogen atoms of the pyridine-containing substituentsare quaternized with (C₁-C₃₀)alkyl groups or with an alkyl group thatcontains one or more ethylene glycol groups.
 2. The polymer of claim 1wherein at least about 10% of the (C₁-C₃₀)alkyl groups are at leastpartially fluorinated.
 3. The polymer of claim 1 further comprising oneor more polymer chain substituents selected from aryl groups, alkylgroups, and alkoxycarbonyl groups, wherein any alkyl, aryl, or alkoxy isoptionally substituted with one or more alkyl, alkoxy, halo,dialkylamino, trifluoromethyl, ethylene glycol, or perfluoroalkylgroups.